Bleomycin for mimicking the effect of ionizing radiations on T cells

ABSTRACT

The present invention relates to a method for mimicking the effects of ionizing radiations on cells, wherein cells are contacted with bleomycin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application pursuant to 35U.S.C. § 371 of International Patent Application PCT/EP2019/064972,filed on Jun. 7, 2019, and published as WO 2019/234229 on Dec. 12, 2019,which claims priority to European Patent Application 18305698.5, filedon Jun. 8, 2018, all of which are incorporated herein by reference intheir entireties for all purposes.

FIELD OF INVENTION

The present invention relates to the determination of the effects ofionizing radiations on cells, in particular on lymphocytes. Morespecifically, the present invention relates to a method for mimickingthe effects of ionizing radiations on cells, without the use of ionizingradiations. The method of the invention may in particular be useful forassessing the individual radiosensitivity of a subject prior to aionizing radiation treatment/radiotherapy treatment.

BACKGROUND OF INVENTION

Treatment by ionizing radiation is one of the leading treatmentmodalities in oncology, and over 50% of patients diagnosed with cancerundergo a treatment by ionizing radiation during their course oftreatment. Although treatment by ionizing radiation is primarily a localtreatment, patients are exposed to a risk of toxicities in the treatmentfield, and in particular in tissues surrounding the tumor, which maydevelop acutely (i.e., in the first 3 months) or late (i.e., more than 3months following the treatment). Severe acute toxicities may have lateconsequences as recovering may be incomplete. In addition, latetoxicities may occur over time and often persist with significantnegative impact on quality of life among cancer survivors.

A number of factors are known to increase the risk of radiation-inducedtoxicity, including individual radiosensitivity (Azria, Betz et al.2012). While toxicity risks for populations of patients are known, thedetermination of an individual's normal tissue radiosensitivity isseldom possible before treatment. Therefore, current practice standardscommonly prescribe radiation dose according to clinical scenarios fromstandard recommendations, without regard to the genotype or phenotype ofthe individual being irradiated.

Previously, a radiosensitivity diagnostic test was developed, based onflow cytometric assessment of RILA (radiation-induced T-lymphocytesapoptosis). This diagnostic test was described as having a clearpotential for selecting individuals likely to display an increasedprobability of toxicity to treatment by ionizing radiation (Ozsahin,Crompton et al. 2005).

However, the RILA test involves a step of treating cells with ionizingradiations. Therefore, the RILA test may not be implemented easily androutinely in standard medical analysis laboratories that are notequipped with cell irradiators, nor authorized to use such devices.

WO 2014/154854 discloses an in vitro method for mimicking the effect ofionizing radiations on T cells (T lymphocytes), comprising contactingsaid T cells with a radiomimetic agent such as bleomycin, but doesn'tdisclose or suggest the durations as herein disclosed.

Ozsahin et al (Clin Cancer Res. 2005 Oct. 15; 11(20):7426-33) disclosethat radiation-induced T-lymphocyte apoptosis can significantly predictdifferences in late toxicity between individuals and could be used as arapid screen for hypersensitive patients to radiotherapy

Weng et al (Mutat Res. 2008 Mar. 29; 652(1):46-53) characterize thedifferential sensitivities of various subpopulations of human whiteblood cells after exposure to H2O2 (an oxidant agent) and bleomycin (aradiomimetic glycopeptide), in vitro by measurement of induced and basalDNA damage.

Adema et al (Int J Radiat Biol. 2003 August; 79(8):655-61) indicate thatbleomycin and radiation give the same sensitivity phenotypes in the G2assay of chromatid breaks (that could be considered as a marker ofradiosensitivity-predisposing genes that respond to DNA damage).However, this test is different from the RILA as described above and theduration of exposure of the cells to bleomycin is different from the oneherein described.

Tedeschi et al (Mutat Res. 2004 Feb. 26; 546(1-2):55-64) describe somekind of genetic basis in the individual expression of induced chromosomedamage following induction in cultured human lymphocytes by in vitrotreatments with aphidicolin (APC) and bleomycin (BLM). Duration ofexposure to bleomycin is different from the one herein disclosed.

Azria et al (Cancer Radiother. 2008 November; 12(6-7):619-24) disclosethat Recently low percentage of CD4 and CD8 lymphocyte apoptosiscorrelate with high grade of sequelae and that patients with severeradiation-induced late side effects possess four or more SNP incandidate genes (ATM, SOD2, TGFB1, XRCC1 et XRCC3) and lowradiation-induced CD8 lymphocyte apoptosis in vitro.

All these documents show that bleomycine can be used, in certainconditions, to generate the same kind of DNA breaks than some radiation.However, these documents are silent as to the capacity of bleomycin tomimic the radiations conditions used for RILA, and as to whether an invitro test can be generated to be able to replace RILA.

Thus, in particular in the field of RILA, there is a need for methodsfor mimicking the effect of a ionizing radiation, without the use of anirradiator (for safety or reglementary reasons).

Therefore, there is an important need to develop an alternative to theRILA test without irradiation step.

The Applicant herein provides such an alternative method, wherein cellsare contacted for a specific time period at a specific concentration ofbleomycin.

SUMMARY

The present invention relates to an in vitro method for mimicking theeffect of a ionizing radiation on T cells, comprising contacting said Tcells with at least one radiomimetic agent, preferably bleomycin, for aperiod of time of at least 50 hours.

In one embodiment, the concentration of said at least one radiomimeticagent, preferably bleomycin, ranges from about 100 to about 300 μg/ml.

In one embodiment, said method mimics the effect of a ionizing radiationat a dose ranging from about 2 to about 10 Gy, preferably from about 6to about 10 Gy, more preferably of about 8 Gy.

In one embodiment, said effect is the percentage of apoptosis oflymphocytes observed 48 hours after irradiation.

In one embodiment, the dose of said at least one radiomimetic agent,preferably bleomycin, is determined using the following mathematicformula

${\overset{\sim}{x}}^{co} = \frac{{\overset{\hat{}}{\beta}}_{0} + {x^{ir}{\overset{\hat{}}{\beta}}_{1}} - {\overset{\hat{}}{\alpha}}_{0}}{{\overset{\hat{}}{\alpha}}_{1}}$

-   -   wherein ({tilde over (x)}^(co)) is the dose of compound to be        applied for mimicking the effect of a specific irradiation dose        (x^(ir)), {circumflex over (β)}₀ corresponds to an apoptosis        average value for irradiation level at zero, {circumflex over        (β)}₁ corresponds to the slope, i.e., to the increase of        apoptosis for each 1 Gy increase, {circumflex over (α)}₀        corresponds to the apoptosis average value for bleomycin        concentration at zero and {circumflex over (α)}₁ corresponds to        the slope, i.e., to the increase of apoptosis for each 1 μg/ml        increase.

In one embodiment, said T cells are contacted with bleomycin at atemperature of about 37° C. and in the presence of about 5% CO₂.

In one embodiment, said T cells are CD8⁺ T cells.

In one embodiment, said T cells are comprised in a T cell containingsample selected from a blood sample, or a sample recovered from bonemarrow, lymph node tissue, cord blood, thymus tissue, tissue from a siteof infection, ascites, pleural effusion, spleen tissue, and tumors,preferably said T cell containing sample is a blood sample, morepreferably said T cell containing sample is a whole blood sample.

In one embodiment, said method is a method for determining theindividual radiosensitivity of a subject, and comprises contacting a Tcell containing sample previously obtained from the subject with atleast one radiomimetic agent, preferably bleomycin for a period of timeof at least 50 hours.

In one embodiment, said subject is diagnosed with cancer.

In one embodiment, said subject is treated or is planned to be treatedwith a ionizing radiation treatment.

In one embodiment, said method is for assessing a risk of developingside effects after a ionizing radiation treatment.

In one embodiment, said method comprises:

-   -   a. contacting a T cell containing sample previously obtained        from a subject with at least one radiomimetic agent, preferably        bleomycin, and    -   b. measuring the percentage of CD8⁺ T cell apoptosis in the        sample from the subject.

In one embodiment, said method comprises comparing the CD8⁺ T cellapoptosis measured at step (b) with a reference CD8⁺ T cell apoptosis.

The present invention further relates to a kit for implementing the invitro method as described hereinabove, comprising means for measuringradiomimetic agent-induced T cell apoptosis. In one embodiment, said kitcomprises at least one radiomimetic agent, preferably bleomycin, andmeans for measuring CD8⁺ T cell apoptosis.

Definitions

In the present invention, the following terms have the followingmeanings:

-   -   The terms “a” and “an” refer to one or to more than one (i.e.,        to at least one) of the grammatical object of the article. By        way of example, “an element” means one element or more than one        element.    -   The term “about” when referring to a measurable value such as an        amount, a temporal duration, and the like, is meant to encompass        variations of ±20% or in some instances ±10%, or in some        instances ±5%, or in some instances ±1%, or in some instances        ±0.1% from the specified value, as such variations are        appropriate to perform the disclosed methods.    -   The term “biochemical marker” refers to a variable that may be        measured in a sample from the subject, said sample being        preferably a blood sample.    -   The term “Cox regression” refers to a usual statistical model        for time-to-event analysis (Cox, et al. 1984). Apart from a        classification algorithm which directly deals with binary or        multi-class outcomes, Cox regression defines a semi-parametric        model to directly relate the predictive variables with the real        outcome, which may be, for example, a survival time (e.g., in        months or years) or a time without occurrence of side effects or        recurrence of a disease. Multivariate Cox function is considered        as the best hazard function in terms of discrimination for        time-to-event endpoint to combine independent parameters.    -   The term “encoding” refers to the inherent property of specific        sequences of nucleotides in a polynucleotide, such as a gene, a        cDNA, or an mRNA, to serve as templates for synthesis of other        polymers and macromolecules in biological processes having        either a defined sequence of nucleotides (e.g., rRNA, tRNA and        mRNA) or a defined sequence of amino acids and the biological        properties resulting therefrom. Thus, a gene, cDNA, or RNA,        encodes a protein if transcription and translation of mRNA        corresponding to that gene, or cDNA, produces the protein in a        cell or other biological system. Both the coding strand, the        nucleotide sequence of which is identical to the mRNA sequence        and is usually provided in sequence listings, and the non-coding        strand, used as the template for transcription of a gene or        cDNA, can be referred to as encoding the protein or other        product of that gene or cDNA.    -   The term “expression” refers to the transcription and/or        translation of a particular nucleotide sequence driven by a        promoter.    -   The term “instructional material” includes a publication, a        recording, a diagram, or any other medium of expression which        can be used to communicate the usefulness of the kit of the        invention. The instructional material of the kit of the        invention may, for example, be affixed to a container which        contains the reagents for implementing the method of the        invention or be shipped together with a container which contains        the reagents for implementing the method of the invention.        Alternatively, the instructional material may be shipped        separately from the container with the intention that the        instructional material be used cooperatively by the recipient.    -   The term “in vitro method” refers to a method comprising steps        performed in vitro (e.g., a measurement of apoptosis) or ex-vivo        (e.g., multivariate cox regression model obtained with apoptosis        percentage, clinical parameters or biochemical marker previously        evaluated on patients).    -   The term “non-invasive”, when referring to a method according to        the present invention, means that the method of the invention        does not comprise obtaining a tissue sample from the body of a        subject. In one embodiment, a blood sample is not considered as        a tissue sample.    -   “ROC” In statistics, a receiver operating characteristic (ROC),        or ROC curve, is a graphical plot that illustrates the        performance of a binary classifier system as its discrimination        threshold is varied. The curve is created by plotting the        sensitivity against the specificity (usually 1−specificity) at        successive values from 0 to 1.    -   “AUROC” stands for area under the ROC curve, and is an indicator        of the accuracy of a prognostic or diagnostic test. ROC curve        and AUROC are well-known in the field of statistics.    -   The term “sensitivity (Se) of a method of prognosis” refers to        the proportion of patients with a risk to develop side-effect        that are correctly identified as such using a method of        prognosis.    -   The term “specificity (Sp) of a method of prognosis” refers to a        measure of the proportion of patients without risk to develop        side-effect that are correctly identified as such using a method        of prognosis.    -   The term “side effect” (or adverse event) refers to an        unfavorable and unintended sign (including an abnormal        laboratory finding), symptom or disease temporally associated        with the use of a medical treatment. In particular, a ionizing        radiation-induced side effect is a side effect induced in a        subject by a ionizing radiation treatment. Severity of side        effects may be defined according to the Common Terminology        Criteria for Adverse Events (CTCAE, e.g., CTCAE v3.0 or CTCAE        v4.0 or CTCAE v5.0). According to the CTCAE, globally the        following grades of side effects may be distinguished: Grade 1:        mild side effect, Grade 2: moderate side effect, Grade 3: severe        side effect, Grade 4: life threatening or disabling side effect        and Grade 5: death related to side effect, but specifically        defined for each symptom. In one embodiment of the invention,        the side effect is at least a Grade 2 side effect. In one        embodiment, the side effect is a Grade 2, 3, 4 or 5        side-effects, preferably a Grade 2, 3 or 4 side-effects.    -   The term “subject” is intended to include any living organisms        (e.g., mammals, preferably humans). In one embodiment, a subject        is a “patient”, i.e., a warm-blooded animal, preferably a human,        who/which is awaiting the receipt of, or is receiving medical        care or was/is/will be the object of a medical procedure, or is        monitored for the development of the targeted disease or        condition. In one embodiment, the subject is an adult (for        example a subject above the age of 18). In another embodiment,        the subject is a child (for example a subject below the age of        18). In one embodiment, the subject is a male. In another        embodiment, the subject is a female.    -   The terms “treat”, “treatment” and “treating” refer to the        reduction or amelioration of the progression, severity and/or        duration of a targeted disease (e.g., cancer), or to the        amelioration of one or more symptoms (preferably, one or more        discernible symptoms) of a targeted disease, wherein said        amelioration results from the administration of one or more        therapies. In one embodiment the terms “treat”, “treatment” and        “treating” refer to the inhibition of the progression of a        targeted disease, either physically by, e.g., stabilization of        at least one discernible symptom, physiologically by, e.g.,        stabilization of a physical parameter, or both. In other        embodiments the terms “treat”, “treatment” and “treating” refer        to the reduction or amelioration of the progression, severity        and/or duration of a targeted disease, or to the amelioration of        one or more symptoms of a targeted disease. A subject is        successfully “treated” for a disease if, after receiving a        therapeutically effective amount of a therapeutic agent or        treatment, the subject shows observable and/or measurable        reduction in the number of pathogenic cells or reduction in the        percent of total cells that are pathogenic; relief to some        extent of one or more of the symptoms associated with the        specific disease; reduced morbidity and mortality, and/or        improvement in quality of life issues. The above parameters for        assessing successful treatment and improvement in the condition        are readily measurable by routine procedures familiar to a        physician.

DETAILED DESCRIPTION

The present invention first relates to an in vitro method for mimickingthe effect of a ionizing radiation on T lymphocytes, comprisingcontacting said T lymphocytes with at least one radiomimetic agent.

The term “radiomimetic agent” as used here refers to a substance thatproduces effects on living cells that are similar to those provoked byionizing radiation.

In one embodiment, the at least one radiomimetic agent is selected fromthe group comprising, but not limited to, bleomycin, streptonigrin,aphidicolin, enediyne antibiotics, and hydrogen peroxide.

In one embodiment, the at least one radiomimetic agent is bleomycin,such as, for example, bleomycin solubilized in ethanol, water or DMSO.Preferably, bleomycin is in an aqueous solvent. Examples of salts ofbleomycin that may be used in the present invention include, but are notlimited to, bleomycin sulfate or bleomycin hydrochloride.

In one embodiment, bleomycin used in the present invention is bleomycinsulfate, preferably in an aqueous solvent. Bleomycin is commerciallyavailable. Examples of bleomycin formulations that may be used include,without limitation, bleomycin provided by Euromedex or by Interchim.

In one embodiment, the T cells are CD4⁺ or CD8⁺ T cells, preferably CD8⁺T cells.

In one embodiment, the T cells of the invention are contained in a Tcell containing sample, or are recovered from a T cell containingsample.

Examples of T cell containing samples include, but are not limited to,whole blood samples and samples recovered from bone marrow, lymph nodetissue, cord blood, thymus tissue, tissue from a site of infection,ascites, pleural effusion, spleen tissue, and tumors. Other T cellcontaining samples can be used in the method herein disclosed, such asan extract containing predominantly PBMC (peripheral blood mononuclearcells) or isolated T cells (in particular isolated CD8 T cells).Predominantly is intended to indicate that at least 80%, more preferablyat least 90% more preferably at least 95% of the cells present in theextract are of the indicated cell type.

In one embodiment, the T lymphocyte containing sample can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as, for example, byleukapheresis.

In one embodiment, said sample is a bodily fluid sample, such as, forexample, a blood, plasma, serum, lymph, urine, cerebrospinal fluid orsweat sample.

In one embodiment, said sample is a blood sample.

In one embodiment, the sample is recovered prior to the implementationof the method of the invention, i.e., the step of recovering the sampleis not part of the method of the invention.

In one embodiment, the T cell containing sample (preferably bloodsample) is collected from a subject in a heparinized tube.

In one embodiment, the T cell containing sample is freshly recovered. Inone embodiment, the T cell containing sample is preserved at roomtemperature (preferably from about 18° C. to about 25° C.) in aheparinized tube.

In another embodiment, the T cell containing sample is cryopreserved(i.e., frozen in liquid nitrogen) and thawed.

In one embodiment, the T cells are cultured in a cell culture mediumprior to the contact with the at least one radiomimetic agent. Examplesof cell culture medium that may be used include, but are not limited to,RPMI-1640 (Thermofisher, France) and DMEM (Dulbecco Modified EagleMedium, Thermofisher). In one embodiment, the cell culture medium isoptionally supplemented with 20% fetal calf serum (FCS) (e.g., FCSprovided by EuroBio, France). In one embodiment, the T cells aremaintained at 37° C. with 5% CO₂.

In one embodiment, the at least one radiomimetic agent is added to theculture medium.

In one embodiment, the T cells are contacted with the at least oneradiomimetic agent for at least about 50 hours, at least about 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79 or at least about 80 hours.

In one embodiment, the T cells are contacted with the at least oneradiomimetic agent for about 50 hours, about 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79 or about 80 hours.

Indeed, the Applicant has demonstrated that, surprisingly, contactingthe T cells with the at least one radiomimetic agent in conditionsmimicking the RILA test, i.e., after 24 hours of cell culture and for atotal duration of about 48 hours, is not sufficient to reproduce theeffect of ionizing radiation on T cells (in particular to obtain thesame apoptosis level in the CD8 lymphocytes as obtained according to theRILA procedure, and in particular when 8 Gy are applied to the cellscultured for 24 hours, and apoptosis is looked at 48 hours afterirradiation). Willing to mimic the effect of ionizing radiation on Tcells, the Applicant further surprisingly demonstrated that a longercontacting step, such as, for example for a period of time ranging fromabout 50 hours to about 80 hours, allows reproducing the effect ofionizing radiation on T cells. In particular, the at least oneradiomimetic agent can be added at the start of the cell culture andmaintained in the medium for at least 50 hours. Advantageously, it ismaintained for at least 60 hours, or for at least or about 72 hours orfor at least or about 75 hours, or for between about 70 and 77 hours. Itis postulated that the apoptosis induction is complex and takes sometime, which is why a longer time of exposition of the cells to theradiomimetic agent is required, even though some markers of radiation(such as DNA breaks) are present in the cells early after the cells areexposed to the radiomimetic agent.

In one embodiment, the T cells are contacted with the at least oneradiomimetic agent at about 37° C. with about 5% CO₂.

In one embodiment, T cells are added in a cell culture medium containingthe at least one radiomimetic agent. In another embodiment, the at leastone radiomimetic agent is added to a culture medium previouslycontaining the T cells.

In one embodiment, the in vitro method of the invention is for mimickingthe effect on T lymphocytes of a ionizing radiation ranging from about 2Gray (Gy) to about 10 Gy, preferably from about 5 to about 9 Gy, morepreferably from about 6 to about 10 Gy, such as, for example, a ionizingradiation of about 6 Gy, about 8 Gy or about 10 Gy.

The skilled artisan would easily understand that the dose of the atleast one radiomimetic agent to be used depend on the ionizing radiationdose to be mimicked.

In one embodiment, said dose is determined according to the followingmathematic formula:

${\overset{\sim}{x}}^{co} = \frac{{\overset{\hat{}}{\beta}}_{0} + {x^{ir}{\overset{\hat{}}{\beta}}_{1}} - {\overset{\hat{}}{\alpha}}_{0}}{{\overset{\hat{}}{\alpha}}_{1}}$wherein {tilde over (x)}^(co) is the dose of compound to be applied formimicking the effect of a specific irradiation dose (x^(ir)),{circumflex over (β)}₀ corresponds to an apoptosis average value forirradiation level at zero, {circumflex over (β)}₁ corresponds to theslope, i.e., to the increase of apoptosis for each 1 Gy increase,{circumflex over (α)}₀ corresponds to the apoptosis average value forbleomycin concentration at zero and {circumflex over (α)}₁ correspondsto the slope, i.e., to the increase of apoptosis for each 1 μg/mLincrease.

In one embodiment, {circumflex over (β)}₀ ranges from about 10 to about20, preferably is of about 14 to 18. In one embodiment, {circumflex over(β)}₁ ranges from about 0.5 to about 2, preferably from about 0.75 toabout 1.25. In one embodiment, {circumflex over (α)}₀ ranges from about7.5 to about 20, preferably from about 10 to about 15. In oneembodiment, {circumflex over (α)}₁ ranges from about 0.03 to about 0.15,preferably from about 0.05 to about 0.1.

In one embodiment, the concentration of the at least one radiomimeticagent ranges from about 50 μg/ml to about 500 μg/ml, preferably fromabout 100 μg/ml to about 300 μg/ml, such as, for example, about 100,125, 150, 175, 200, 225, 250, 275, or 300 μg/ml.

In one embodiment, the at least one radiomimetic agent is bleomycin andthe concentration of bleomycin is ranging from about 50 μg/ml to about500 μg/ml, preferably from about 100 μg/ml to about 300 μg/ml, such as,for example, about 100, 125, 150, 175, 200, 225, 250, 275, or 300 μg/ml.

In one embodiment, the method of the invention is for mimicking theeffect of a 6 Gy irradiation, and the concentration of the at least oneradiomimetic agent (preferably of bleomycin) ranges from about 50 μg/mlto about 500 μg/ml, preferably from about 100 μg/ml to about 300 μg/ml,such as, for example, about 100, 125, 150, 175, 200, 225, 250, 275, or300 μg/ml.

In one embodiment, the method of the invention is for mimicking theeffect of a 8 Gy irradiation, and the concentration of the at least oneradiomimetic agent (preferably of bleomycin) ranges from about 50 μg/mlto about 500 μg/ml, preferably from about 100 μg/ml to about 300 μg/ml,such as, for example, about 100, 125, 150, 175, 200, 225, 250, 275, or300 μg/ml. In one embodiment, the method of the invention is formimicking the effect of a 8 Gy irradiation, and the concentration of theat least one radiomimetic agent (preferably of bleomycin) ranges fromabout 135 to about 160 μg/mL.

In one embodiment, the method of the invention is for mimicking theeffect of a 10 Gy irradiation, and the concentration of the at least oneradiomimetic agent (preferably of bleomycin) ranges from about 50 μg/mlto about 500 μg/ml, preferably from about 100 μg/ml to about 300 μg/ml,such as, for example, about 100, 125, 150, 175, 200, 225, 250, 275, or300 μg/ml.

In one embodiment, the method of the invention comprises:

-   -   a) contacting T cells with at least one radiomimetic agent, and    -   b) measuring radiomimetic agent-induced T-cell apoptosis.

In one embodiment, the method of the invention comprises:

-   -   a) contacting a T cell containing sample with at least one        radiomimetic agent, and    -   b) measuring radiomimetic agent-induced T-cell apoptosis in the        T cell containing sample.

In one embodiment, the in vitro method of the invention comprisesmeasuring radiomimetic agent-induced apoptosis of CD4 and/or CD8T-lymphocyte. In one embodiment, the method of the invention comprisesmeasuring radiomimetic agent-induced CD8 T-lymphocyte apoptosis.

In one embodiment, at the end of the contacting step, T cells aretransferred to pre-labeled centrifuge tubes, for centrifugation, such asfor example, for 5 minutes at 390 g. After centrifugation, the samplemay be labeled with a fluorochrome coupled-anti-CD4 and/or fluorochromecoupled-anti-CD8 antibody.

Then, in one embodiment, a lysis buffer is added to the centrifuge tubein order to lyse any non-lymphocyte cell (such as, for example, redblood cells). An example of lysis buffer is the ammonium chloride basedlysing reagent provided by Beckton Dickinson (USA).

After lysis, reagents are added to the tube for evaluating lymphocytesapoptosis according to usual methods known from the person skilled inthe art.

Examples of methods for measuring T-lymphocytes (preferably CD8T-lymphocytes) apoptosis include, but are not limited to, FACS analysis(e.g., with propidium iodide and RNase A as reagents), dosage of AnnexinV, and dosage of caspases. Preferably the evaluation of T lymphocytesapoptosis is carried out by FACS analysis.

In one embodiment, the method of the invention comprises the measurementof apoptosis features occurring in the cell such as, for example,membrane asymmetry (that may be visualized, for example, by thephosphatidyl serine externalization), membrane permeability,mitochondria metabolic activity, caspase activation and chromatincondensation.

Thus, in one embodiment, the apoptosis measurement step of the inventioncomprises the use of specific reagents to evaluate T cell apoptosisfeatures. Examples of such reagents include, but are not limited to,propidium iodide, 7-AAD, fluorochrome coupled-annexin, YO-PRO dyes,PO-PRO dyes, Resazurin, Hoechst, fluorochrome coupled-caspase antibodiesand JC-1 dye. Preferably, the method of the invention uses propidiumiodide.

In one embodiment, the method of the invention comprises measuring apercentage of apoptotic cells after the contacting step.

In one embodiment, the measurement of apoptosis is carried out intriplicate.

In one embodiment, the method of the invention further comprises a stepof measuring a percentage of apoptotic T cells in a control sample notcontacted with the at least radiomimetic agent (“basal T cellapoptosis”). In one embodiment, for measuring basal T cell apoptosis,cells are kept in the exact same conditions than the test sample (e.g.,medium, temperature, CO₂, etc. . . . ), except that these cells are notcontacted with the at least one radiomimetic agent.

In one embodiment, the in vitro method of the invention allowsdetermining the individual radiosensitivity of a subject.

In one embodiment, the in vitro method of the invention is thus forassessing the risk of developing side effects after ionizing radiationin a subject.

In one embodiment, the method of the invention aims at predicting therisk of developing side effects during ionizing radiation treatment andduring a period of about 1, 3, 6, 12, 18, 24, 30 or 36 months afterionizing radiation treatment.

In one embodiment, the in vitro method of the invention aims atpredicting the risk of developing acute side effects, i.e., side effectsoccurring during ionizing radiation treatment or less than about 1 week,2 weeks, 3 weeks or 4 weeks after ionizing radiation treatment, or lessthan about 1, 2 or 3 months after ionizing radiation treatment.

In another embodiment, the in vitro method of the invention aims atpredicting the risk of developing late side effects, i.e., side effectsoccurring at least about 3 months after ionizing radiation treatment,such as, for example, between about 3 months and about 6 months afterionizing radiation treatment, between about 3 months and about 12 monthsafter ionizing radiation treatment, between about 3 months and about 18months after ionizing radiation treatment, between about 3 months andabout 2 years after ionizing radiation treatment, between about 3 monthsand about 30 months after ionizing radiation treatment, or between about3 months and about 3 years after ionizing radiation treatment. In oneembodiment, the late side effects occur about 4, 5, 6, 7, 8, 9, 10, 11,12 months or more after ionizing radiation treatment, or 2 or 3 years ormore after ionizing radiation treatment.

In one embodiment, the side-effects as listed hereinabove are at leastGrade 2 side effects according to the CTCAE, e.g., to the v3.0 CTCAE,the v4.0 CTCAE or the v5.0 CTCAE. In one embodiment, the side-effects aslisted hereinabove are Grade 2, Grade 3, Grade 4 or Grade 5 side effectsaccording to the CTCAE, e.g., to the v3.0 CTCAE, preferably Grade 2,Grade 3, or Grade 4.

In one embodiment, the method of the invention is non-invasive.

In one embodiment, the method of the invention comprises contacting Tcells previously obtained from the subject with at least oneradiomimetic agent.

In one embodiment, the subject is a human.

In one embodiment, the subject is, was or will be treated by ionizingradiation.

In one embodiment, the T cells or T cell containing sample are/isobtained from the subject before the beginning of the treatment withionizing radiation.

In one embodiment, the subject is diagnosed with a tumor. In oneembodiment, the subject is diagnosed with a malignant tumor. In anotherembodiment, the subject is diagnosed with a non-malignant (or benigntumor). Examples of non-malignant tumor include, but are not limited to,moles, uterine fibroids, neoplasms (e.g., lipoma, chondroma, adenoma,teratoma, hamartoma and the like).

In another embodiment, the subject is diagnosed with a non-malignantdisorder that may be treated by ionizing radiations. Examples ofnon-malignant disorders that may be treated by ionizing radiationsinclude, but are not limited to, Graves' disease, calcaneal spur andkeloids.

In one embodiment, the subject is diagnosed with cancer. Examples ofcancers include, but are not limited to, prostate cancers, breastcancers, gastrointestinal cancers (e.g., colon cancer, small intestinecancer or colorectal cancer), stomach cancers, pancreas cancers, lungcancers (e.g., non-small cell lung cancer), mesothelioma, bladdercancers, kidney cancers, thyroid cancers, cardiac cancers, genitourinarytract cancers, liver cancers, bone cancers, nervous system cancers(e.g., brain cancer), gynecological cancers (e.g., ovarian cancer),testicular cancer, hematologic cancers, throat cancers, head and neckcancers, oral cancers, skin cancers, and adrenal glands cancers.

In one embodiment, said cancer is a tumor, such as, for example, a solidtumor. In another embodiment, said cancer is a blood cancer. In anotherembodiment, said cancer is a hematologic malignancy.

Examples of breast cancer include, but are not limited to ductalcarcinoma in situ, invasive ductal carcinoma, tubular carcinoma of thebreast, medullary carcinoma of the breast, mucinous carcinoma of thebreast, papillary carcinoma of the breast, cribriform carcinoma of thebreast, invasive lobular carcinoma, inflammatory breast cancer, lobularcarcinoma in situ, male breast cancer, Paget's disease of the nipple,phyllodes tumors of the breast and recurrent & metastatic breast cancer.

Examples of gastrointestinal cancer include, but are not limited to,esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoidtumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma), colon, colorectal, and rectal cancers.

Examples of lung cancer include, but are not limited to, adenocarcinoma(formerly bronchioloalveolar carcinoma), undifferentiated small cellcarcinoma, undifferentiated large cell carcinoma, small cell carcinoma,large cell carcinoma, large cell neuroendocrine tumors, small cell lungcancer (SCLC), undifferentiated non-small cell lung cancer, bronchialadenoma, sarcoma, lymphoma, chondromatosis hamartoma, Pancoast tumorsand carcinoid tumors.

Examples of mesothelioma include, but are not limited to, pleuralmesothelioma, peritoneal mesothelioma, pericardial mesothelioma, endstage mesothelioma as well as epithelioid, sarcomatous, and biphasicmesothelioma.

Examples of bladder cancer include, but are not limited to, transitionalcell bladder cancer (formerly urothelial carcinoma), invasive bladdercancer, squamous cell carcinoma, adenocarcinoma, non-muscle invasive(superficial or early) bladder cancer, sarcomas, small cell cancer ofthe bladder and secondary bladder cancer.

Examples of cardiac cancer include, but are not limited to, sarcoma(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,rhabdomyoma, fibroma, lipoma and teratoma.

Examples of genitourinary tract cancer include, but are not limited to,kidney (adenocarcinoma, Wihn's tumor [nephroblastoma], lymphoma,leukemia), bladder and urethra (squamous cell carcinoma, transitionalcell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), andtestis cancers (seminoma, teratoma, embryonal carcinoma,teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma,fibroma, fibroadenoma, adenomatoid tumors, lipoma).

Examples of liver cancer include, but are not limited to, hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, and hemangioma.

Examples of bone cancers include, but are not limited to, osteogenicsarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochondroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors.

Examples of nervous system cancers include, but are not limited to,skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitisdeformans), meninges cancer (meningioma, meningosarcoma, gliomatosis),and brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma).

Examples of gynecological cancers include, but are not limited to,uterus cancer (endometrial carcinoma), cervix cancer (cervicalcarcinoma, pre-tumor cervical dysplasia), ovaries cancer (ovariancarcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma,unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydigcell tumors, dysgerminoma, malignant teratoma), vulva cancer (squamouscell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma,melanoma), and vagina cancer (clear cell carcinoma, squamous cellcarcinoma, botryoid sarcoma [embryonal rhabdomyosarcoma], fallopiantubes cancer [carcinoma]).

Examples of hematologic cancers include, but are not limited to, bloodcancer (myeloid leukemia [acute and chronic], acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma [malignant lymphoma].

Examples of skin cancers include, but are not limited to, malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, andkeloids.

Examples of adrenal glands cancers include, but are not limited to,neuroblastoma.

In one embodiment, said cancer is advanced cancer. As used herein, theterm “advanced cancer” refers to a cancer that has spread to otherplaces in the body and usually cannot be cured or controlled withtreatment. In particular, locally advanced cancer is cancer that hasspread from where it started to nearby tissue or lymph nodes.

In one embodiment, said cancer is an unresectable cancer. As usedherein, the term “unresectable cancer” refers to a cancer that may notbe removed by surgery.

In one embodiment, said cancer is a recurrent cancer. As used herein,the term “recurrent cancer” refers to a cancer that has recurred (comeback). The cancer may come back to the same place as the original(primary) tumor or to another place in the body.

In one embodiment, said cancer is a metastatic cancer. As used herein,the term “metastatic cancer” refers to a cancer that has spread from theplace where it first started to another place in the body. A tumorformed by metastatic cancer cells may be called a metastatic tumor or ametastasis. The metastatic tumor contains cells that are like those inthe original (primary) tumor.

In one embodiment, the subject is planned to be treated by ionizingradiation, and the method of the invention is implemented before thebeginning of the treatment by ionizing radiation.

The term “ionizing radiation”, as used herein, refers to a treatmentinvolving the use of radiation such as, for example, X-rays (electron orphoton beams), gamma rays, or protons, to kill or damage cancer or tumorcells and stop them from growing and multiplying.

In one embodiment, the ionizing radiation involves the use of X-rays.

In one embodiment, the side effects are side effects induced by ionizingradiation in the area of the treatment, such as, for example, in thepelvic area for the treatment of prostate cancer, or in the breast areafor the treatment of breast cancer.

Examples of ionizing radiation-induced side effects that may be inducedduring the treatment of prostate cancer include, but are not limited to,genitourinary (such as, for example, urinary and sexual toxicities),gastrointestinal and neurologic toxicities.

Examples of ionizing radiation-induced side effects that may be inducedduring the treatment of breast cancer include, but are not limited to,atrophic skin, telangiectasia, induration (fibrosis), necrosis orulceration. In one embodiment, the ionizing radiation-induced sideeffects are breast late side effects, i.e., side effects induced duringthe treatment of breast cancer (e.g., atrophic skin, telangiectasia,induration (fibrosis), necrosis or ulceration) and occurring at leastabout 3 months after ionizing radiation treatment (such as, for example,between about 3 months and about 6 months after ionizing radiationtreatment, between about 3 months and about 12 months after ionizingradiation treatment, between about 3 months and about 18 months afterionizing radiation treatment, between about 3 months and about 2 yearsafter ionizing radiation treatment, between about 3 months and about 30months after ionizing radiation treatment, or between about 3 months andabout 3 years after ionizing radiation treatment or about 4, 5, 6, 7, 8,9, 10, 11, 12 months or more after ionizing radiation treatment, or 2 or3 years or more after ionizing radiation treatment).

In one embodiment, the in vitro method of the invention furthercomprises a step of comparing the radiomimetic agent-induced T cell witha reference radiomimetic agent-induced T cell apoptosis, therebydetermining if the subject presents a high or low risk to developionizing radiation-induced side-effects.

In one embodiment, the reference radiomimetic agent-induced T cellapoptosis corresponds to the apoptosis measured in a referencepopulation. In one embodiment, the reference apoptosis was measured in areference population comprising patients (e.g., cancer patients) treatedwith ionizing radiation and having experienced radiation-inducedside-effects during follow-up (such as, for example, during ionizingradiation and/or during the follow-up after ionizing radiation, such as,for example, 1 month, 3, 6, 12, 18, 24, 30 or 36 months after the end ofthe ionizing radiation). In another embodiment, the reference apoptosiswas measured in a reference population comprising patients (e.g., cancerpatients) treated with ionizing radiation and having experienced noionizing radiation-induced side-effects during follow-up (such as, forexample, during ionizing radiation and/or during the follow-up afterionizing radiation, such as, for example, 1 month, 3, 6, 12, 18, 24, 30or 36 months after the end of the ionizing radiation).

A reference apoptosis can be derived from population studies, includingwithout limitation, such subjects having similar age range, subjects inthe same or similar ethnic group, similar disease history, similarionizing radiation treatment and the like.

In one embodiment, the reference apoptosis is constructed usingalgorithms and other methods of statistical and structuralclassification.

In one embodiment, the reference apoptosis corresponds to the meanapoptosis measured in the reference population. In one embodiment of theinvention, the reference apoptosis corresponds to the median apoptosismeasured in the reference population.

In one embodiment, the method of the invention is computerized (orcomputer-implemented).

In one embodiment, the method of the invention comprises determining ifthe radiomimetic agent-induced T cell apoptosis is superior to thereference apoptosis, or inferior or equal to said reference apoptosis.

In another embodiment, the method of the invention comprises determiningthe percentile wherein the radiomimetic agent-induced T cell apoptosismeasured for the subject may be positioned. According to thisembodiment, the radiomimetic agent-induced T cell apoptosis valuesmeasured in a reference population are classified in percentiles,wherein the radiomimetic agent-induced T cell apoptosis values obtainedfor all subjects of the reference population are ranged according totheir numerical value in ascending order. In one embodiment of theinvention, the percentiles are percentiles of subjects, i.e., eachpercentile comprises the same number of subjects. Therefore, the firstpercentile corresponds to subjects with the lowest radiomimeticagent-induced T cell apoptosis values, while the last percentilecorresponds to subjects with the highest radiomimetic agent-induced Tcell apoptosis values. In one embodiment, when three percentiles aredrawn, each percentile is named a tertile. In another embodiment, whenfour percentiles are drawn, each percentile is named a quartile. Inanother embodiment, when five percentiles are drawn, each percentile isnamed a quintile.

The skilled artisan knows how to determine the reference apoptosis orpercentiles of radiomimetic agent-induced T cell apoptosis values fromradiomimetic agent-induced T cell apoptosis values obtained in areference population.

A non-limiting example of such method include the drawing of a ROC curveto determine the cut-off of radiomimetic agent-induced T cell apoptosisvalue measured for the subject with side effect vs subject without sideeffect (AUROC) which maximize Se and Sp.

In one embodiment, determining the radiomimetic agent-induced T cellapoptosis, will help the physician to adapt the dose and sequences ofionizing radiation treatment to the patient to limit theionizing-radiation induced side effects, and optionally to adapt thetreatment by replacing ionizing radiation treatment with othertherapeutic treatments.

In a particular embodiment, the radiomimetic agent-induced T cellapoptosis is used to choose a suitable treatment for the patient, suchas an appropriate ionizing radiation regimen.

Another object of the invention is a method for implementing an adaptedpatient care (or treatment) for a patient, wherein said methodcomprises:

-   -   assessing the risk for said patient to develop ionizing        radiation-induced side-effects, using the in vitro method as        described hereinabove;    -   implementing an adapted patient care (or treatment) depending on        the risk for the patient to develop ionizing radiation-induced        side-effects.

In one embodiment, the patient presents a high risk to develop ionizingradiation-induced side effects and the adapted patient care may beselected from the group comprising decreased ionizing radiation dosageregimen, or alternative treatment, such as, for example, surgery.

In one embodiment, the patient presents no or only a low risk to developionizing radiation-induced side effects and the adapted patient care maybe selected from the group comprising increased ionizing radiationdosage regimen.

Another object of the present invention is thus a computer software forimplementing the method of the invention.

In one embodiment, the in vitro method of the invention is implementedwith a microprocessor comprising a software configured to calculate aradiomimetic agent-induced T cell apoptosis.

Another object of the present invention is directed to a systemincluding a machine-readable memory, such as a computer and/or acalculator, and a processor configured to compute said mathematicalfunction, in particular said multivariate Cox function. This system maybe dedicated to perform the method according to the invention.

Another object of the present invention is a kit for implementing themethod of the present invention, wherein the kit comprises reagents formeasuring radiomimetic agent-induced T cell apoptosis, as defined in thepresent invention.

Another object of the present invention is a kit for implementing themethod of the present invention, wherein the kit comprises:

-   -   a box/container and bag suited for biological transportation of        a T cell containing sample, in particular a blood sample, and        optionally reagents for isolating T cells; and    -   reagents for measuring radiomimetic agent-induced T cell        apoptosis.

Another object of the present invention is thus a kit for detecting therisk of developing a ionizing radiation-induced side effect in a subjectusing the method of the present invention, wherein said kit comprises:

-   -   a box/container and bag suited for biological transportation of        a T cell containing sample, in particular a blood sample, and        optionally reagents for isolating T cells    -   reagents for determining radiomimetic agent-induced T lymphocyte        apoptosis as defined in the present invention.

By “kit” is intended any manufacture (e.g., a package or a container).The kit may be promoted, distributed, or sold as a unit for performingthe methods of the present invention. Furthermore, any or all of the kitreagents may be provided within containers that protect them from theexternal environment, such as in sealed containers. The kits may alsocontain an instructional material describing the kit and methods for itsuse. Kits are also provided that are useful for various purposes. Thelabel or instructional material may provide a description of the contentof the kit as well as instructions for the intended use.

In one embodiment, the reagents for determining radiomimeticagent-induced T lymphocyte apoptosis as defined in the present inventioncorrespond to some or all specific reagents required to:

-   -   run the step of culture of T cells as described hereinabove, in        the presence of at least one radiomimetic agent, such as, for        example, a culture medium suitable for culturing T cells (such        as, for example, RPMI supplemented with 20% FCS), and said        radiomimetic agent (such as, for example, bleomycin);    -   run the apoptosis measurement assay (for example, using a flow        cytometer), such as, for example, propidium iodide, fluorochrome        coupled-annexin, YO-PRO dyes, PO-PRO dyes, Resazurin, Hoechst,        fluorochrome coupled-caspases.

In one embodiment, the in vitro method of the invention is a method forassessing the individual radiosensitivity of a subject with a breastcancer (preferably for determining the risk of developing ionizingradiation-induced breast late side effects), and the method of theinvention comprises:

-   -   a) contacting a T cell containing sample previously obtained        from a subject, with at least one radiomimetic agent;    -   b) measuring a radiomimetic agent-induced T lymphocyte apoptosis        in the sample;    -   c) determining the level of at least two clinical parameters in        the subject,    -   d) optionally measuring at least one biochemical marker in the        subject, and    -   e) optionally combining in a mathematical function, the        radiomimetic agent-induced T cell apoptosis measured in step b)        with said at least two clinical parameters determined at step        c), and optionally with the at least one biochemical marker        measured at step d).

By “clinical parameter” it is meant any clinical parameter related tothe subject and relevant to assess an increased risk of ionizingtreatment-induced toxicity in said subject. Examples of clinicalparameters include, but are not limited to, age, breast volume, adjuvanthormonotherapy, boost (complement dose of irradiation), nodeirradiation, and tobacco smoking. Preferably, the at least two clinicalparameters measured at step c) comprise tobacco smoking habits andadjuvant hormonotherapy. In one embodiment, the method of the inventioncomprises determining in step (c) if the subject previously received oris currently receiving an adjuvant hormonotherapy. As used herein, theterm “adjuvant hormonotherapy” refers to a treatment started after orconcomitantly or before surgery, chemotherapy, and/or ionizing radiationtherapy to lower the risk of recurrence of the cancer.

Hormone receptor-positive breast cancer depends on hormones calledestrogen and/or progesterone to grow. Adjuvant hormonotherapy allows tolower the levels of these hormones in the body or to block the hormonesfrom getting to any remaining cancer cells.

Examples of adjuvant hormonotherapy that may be used for the treatmentof breast cancer include, but are not limited to, tamoxifen, aromataseinhibitors (AIs), such as anastrozole (Arimidex) and letrozole (Femara),exemestane (Aromasin), and ovarian suppression by surgery or by drugsselected from gonadotropin, luteinizing, goserelin (Zoladex) andleuprolide (Lupron).

In one embodiment, the method of the invention comprises determining instep (c) the tobacco smoking habits of the subject.

As used herein, the terms “determining the tobacco smoking habits of thesubject” means determining if the subject is a tobacco smoking subject(either daily smoker, intermittent smoker or non-daily smoker) or anon-smoking subject as defined hereafter.

A “daily smoker” may be defined as a subject that is currently smokingon a daily basis. A “intermittent smoker” may be defined as a subjectnot smoking on a daily basis (DiFranza et al., 2007; Lindstrom,Isacsson, & the Malmo Shoulder-Neck Study Group, 2002) or smoking on1-15 days in the previous month (McCarthy, Zhou, & Hser, 2001). A“non-daily smoker” may be defined as a subject (i) smoking at leastweekly (but not daily) or less often than weekly; (ii) smoking at least100 cigarettes in the lifetime and currently smoking some days; (iii)smoking more than 100 cigarettes in the lifetime, currently smoking somedays, and having smoked less than 30 cigarettes during the past 30 days;(iv) smoking more than 100 cigarettes in the lifetime and having smokedsome days or 1-2 days in the previous 30 days; or (v) smoking fewer than100 cigarettes in the lifetime and having smoked in the previous 30 days(Gilpin, White, & Pierce, 2005; Hassmiller et al., 2003; Husten,McCarty, Giovino, Chrismon, & Zhu, 1998; Leatherdale, Ahmed, Lovato,Manske, & Jolin, 2007; McDermott et al., 2007; Tong, Ong, Vittinghoff, &Perez-Stable, 2006; Wortley, Husten, Trosclair, Chrismon, & Pederson,2003).

A “some-day smoker” may be defined as a subject having ever smoked 100cigarettes during the smoker's lifetime and currently smoking on somedays (not every day; CDC, 1993; Hassmiller, Warner, Mendez, Levy, &Romano, 2003).

A “never daily smoker” may be defined as a subject having never smokeddaily for 6 months or more (Gilpin et al., 1997).

In one embodiment, the method of the invention comprises measuring atstep d) at least one biochemical marker. In one embodiment, said atleast one biochemical marker is selected from the group comprisingproteins of individual radiosensitivity and genes of individualradiosensitivity.

In one embodiment, said at least one biochemical marker is a protein ofindividual radiosensitivity, preferably selected from the groupconsisting of AK2 (adenylate kinase 2), HSPA8 (Heat shock cognateprotein 71 kDa, also referred to as HSC70), ANX1 (Annexin 1), APEX1(DNA-(apurinic or apyrimidinic site) lyase) and IDH2 (mitochondrialisocitrate dehydrogenase 2), fragments and combinations thereof.

In one embodiment, said at least one biochemical marker is a combinationof at least two proteins of individual radiosensitivity, of at leastthree proteins of individual radiosensitivity, of at least four proteinsof individual radiosensitivity, or of five proteins of individualradiosensitivity, preferably selected from the group consisting of AK2,HSPA8, ANX1, APEX1 and IDH2.

As used herein, a “protein of individual radiosensitivity” refers to aprotein whose expression (either at the protein or RNA level) isindicative of the individual radiosensitivity of the subject.

Consequently, in one embodiment, measuring at least one biochemicalmarker at step (c) corresponds to measuring a protein level of at leastone protein of individual radiosensitivity in a sample from the subjector to measuring a nucleic acid encoding said protein in a sample fromthe subject. In one embodiment, in a first step, proteins and/or nucleicacids are isolated from a biological sample previously obtained from thesubject. A method according to the invention may thus include protein ornucleic acid extraction, purification and characterization, using wellknown biochemistry methods. The presence or level of said protein ofindividual radiosensitivity may be determined by methods well known inthe art. Examples of such methods include, but are not limited to, amethod based on immune-detection, a method based on western blot, amethod based on mass spectrometry, a method based on chromatography, ora method based on flow cytometry, and a method for specific nucleic aciddetection. Specific examples of in vitro methods for determining aprotein level in a sample are well-known in the art, and include, butare not limited to, immunohistochemistry, Multiplex methods (Luminex),western blot, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA,fluorescent-linked immunosorbent assay (FLISA), enzyme immunoassay(EIA), radioimmunoassay (RIA), flow cytometry (FACS) and the like.

In one embodiment, the presence and level of expression of proteins canbe determined directly or be analyzed at the nucleic level by detecting,and preferably quantifying, protein-specific nucleic acids, andparticularly mRNA (i.e., assessing the transcription level of theprotein). Methods for assessing the transcription level of a protein arewell known in the prior art. Examples of such methods include, but arenot limited to, RT-PCR, RT-qPCR, Northern Blot, hybridization techniquessuch as, for example, use of microarrays, and combination thereofincluding but not limited to, hybridization of amplicons obtained byRT-PCR, sequencing such as, for example, next-generation DNA sequencing(NGS) or RNA-seq (also known as “Whole Transcriptome ShotgunSequencing”) and the like.

In one embodiment, said at least one biochemical marker is a gene ofindividual radiosensitivity, preferably selected from the groupconsisting of TGFβ, SOD2, TNFα, and XRCC1.

As used herein, a “gene of individual radiosensitivity” refers to a genewhose expression (either at the protein or RNA level) is indicative ofthe individual radiosensitivity of the subject, or to a gene comprisingat least one single nucleotide polymorphism (SNP) indicative of theindividual radiosensitivity of the subject, or involved in the fibrosispathway and ROS management.

Consequently, in one embodiment, measuring at least one biochemicalmarker at step (c) corresponds to measuring an expression level of ordetermining the presence of a SNP in at least one gene of individualradiosensitivity in a sample from the subject.

The presence or level of expression of said gene of individualradiosensitivity may be determined by a usual method known from manskilled in the art. A non-limiting list of such methods is shownhereinabove.

In one embodiment, the in vitro method for determining presence or levelof expression of a gene of individual radiosensitivity is as disclosedin Azria et al., 2008 and includes lymphocyte isolation, DNA extractionand amplification, and denaturating high-performance liquidchromatography or the Surveyor nuclease assay using a Transgenomic WAVEHigh Sensitivity Nuclei Acid Fragment Analysis System.

In one embodiment, the in vitro method of the invention comprisesmeasuring at step (c) at least one protein of individualradiosensitivity and at least one gene of individual radiosensitivity.

In one embodiment, the in vitro method of the invention comprises:

-   -   a) contacting a T cell containing sample previously obtained        from a subject, with at least one radiomimetic agent;    -   b) measuring a radiomimetic agent-induced T lymphocyte apoptosis        in the sample;    -   c) determining if the subject is/was treated with an adjuvant        hormonotherapy, and determining the tobacco smoking habits of        the subject,    -   d) optionally measuring at least one biochemical marker in the        subject, and    -   e) optionally combining in a mathematical function, the        radiomimetic agent-induced T cell apoptosis measured in step b)        with said at least two clinical parameters determined at step        c), and optionally with the at least one biochemical marker        measured at step d).

In one embodiment, the method of the invention comprises at step e) astep of combining the radiomimetic agent-induced T cell apoptosismeasured in step (b), the at least two clinical parameters measured atstep c) (preferably presence or absence of an adjuvant hormonotherapy,and tobacco smoking habits) and optionally the at least one biochemicalparameter measured in step d) in a mathematical function, therebyobtaining an end-value.

In one embodiment, said mathematical function is a multivariate analysisusing a binary logistic regression, a multiple linear regression or anytime-dependent regression.

In one embodiment, said mathematical function is a Cox proportionalhazard regression model.

In one embodiment, for the clinical parameters as listed hereinabove andthat may be present or absent, the presence of said parameter is givenvalue=1 in the mathematical function, preferably in the multivariate Coxfunction, while its absence is given value=0 in the mathematicalfunction, preferably in the multivariate Cox function.

For example, in one embodiment, at step (c), if the subject is treatedby an adjuvant hormonotherapy, the value “1” is affected. In oneembodiment, at step (c), if the subject is not treated by an adjuvanthormonotherapy, the value “0” is affected to the subject.

In one embodiment, for the clinical parameter “tobacco smoking”, atobacco smoking patient is defined consistently as daily smoker,intermittent smoker or non-daily smoker (and given value=1 in themathematical function, preferably in the multivariate Cox function),while a non-smoking patient is defined as some-day smoker or never dailysmoker (and given value=0 in the mathematical function, preferably inthe multivariate Cox function).

In one embodiment, for the clinical parameter “age”, the median age is55 years to define patients being 55 years old or less (and givenvalue=0 in the mathematical function, preferably in the multivariate Coxfunction) and patients being older than 55 years old (and given value=1in the mathematical function, preferably in the multivariate Coxfunction).

In another embodiment, for continued data (in particular for thebiochemical markers optionally measured at step d) and for theradiomimetic agent-induced T cell apoptosis measured at step b)), theparameter is given its exact value in the mathematical function,preferably in the multivariate Cox function.

In one embodiment, the mathematical function of the invention is abinary logistic regression, a multiple linear regression or anytime-dependent regression.

In one embodiment, the end-value is obtained by combining the measuresof radiomimetic agent-induced T lymphocyte apoptosis, at least twoclinical parameters (preferably adjuvant hormonotherapy and tobaccosmoking habits), and optionally at least one biochemical marked, in aregression formula established using multivariate analysis.

In one embodiment, said formula is expressed as:

A+B1*(radiomimetic agent-induced T lymphocyte apoptosis)+B2*(firstclinical parameter, preferably adjuvant hormonotherapy)+B3*(secondclinical parameter, preferably tobacco smoking habits)+ . . . +Bn*((n−1)clinical parameter or biochemical marker), wherein A, B1, B2, . . . , Bnare predetermined coefficients.

In another embodiment, said formula is expressed as:

C+[D1*LN(radiomimetic agent-induced T lymphocyteapoptosis)]+[D2*LN(first clinical parameter, preferably adjuvanthormonotherapy)]+[D3*LN(second clinical parameter, preferably tobaccosmoking habits)]+ . . . +[Dn*LN((n−1) clinical parameter or biochemicalmarker)], wherein C, D1, D2, . . . Dn are predetermined coefficients.

In another embodiment, said formula is expressed as:

E+exp[F1*(radiomimetic agent-induced T lymphocyte apoptosis)]+[F2*(firstclinical parameter, preferably adjuvant hormonotherapy)]+[F3*(secondclinical parameter, preferably tobacco smoking habits)]+ . . .+[Fn*((n−1) clinical parameter, or biochemical marker)], wherein E, F1,F2, . . . , Fn are predetermined coefficients.

In one embodiment, the regression is a multivariate Cox regression.

In one embodiment, said regression is time-dependent, preferably is atime-dependent multivariate regression.

In one embodiment, said regression is a multivariate time-related model,preferably a Cox proportional hazard regression model.

In one embodiment of the present invention, the independent parameterscombined in the Cox regression are radiomimetic agent-induced Tlymphocyte apoptosis, adjuvant hormonotherapy, tobacco smoking habitsand optionally at least one biochemical markers. In one embodiment ofthe present invention, the independent parameters combined in the Coxregression are radiomimetic agent-induced T lymphocyte apoptosis,adjuvant hormonotherapy, and tobacco smoking habits.

The multivariate Cox function may usually be obtained by combining therelative weight of each parameter, as individually determined in themultivariate Cox regression, with a negative sign when the markersharbor a negative correlation with the observation of breast late sideeffect.

In one embodiment, in order to define the multivariate Cox model of theinvention (i.e., “modelling”), a classification of breast cancerpatients is made based on the detection of ionizing radiation-inducedside effects, preferably of breast late side effects as describedhereinabove, during the clinical follow-up of studies.

In one embodiment, said modelling may be based on a population (e.g., amulticenter population) of breast cancer patients treated by ionizingradiation (which may be named “reference population”). The steps tobuild up the model may thus consist in:

-   -   the measurement of the percentage of radiomimetic agent-induced        T lymphocyte apoptosis in all the subjects of the population;    -   the identification of biochemical markers (e.g, proteins and/or        genes of individual radiosenstivity) and of clinical parameters        relevant to assess an increased risk of ionizing radiation        toxicity in a subject;    -   the use of said identified biochemical markers and clinical        parameters in a multicenter clinical trial to identify relevant        variables as prognostic factors of ionizing radiation-induced        side effects, in particular breast late side effects, i.e., as        variables that are indicative of a specific risk to develop        ionizing radiation-induced side effects, in particular breast        late side effects;    -   the application of these variables on the large multicenter        clinical trial to identify the predictive role of the        combination of the radiomimetic agent-induced T lymphocyte        apoptosis, biochemical markers (e.g, proteins and/or genes of        individual radiosensitivity) and of clinical parameters for        developing ionizing radiation-induced side effects, in        particular breast late side effects.

By “multicenter research trial” it is meant a clinical trial conductedat more than one medical center or clinic.

In one embodiment, the multivariate Cox model is:

Hazard (experiencing a ionizing radiation-induced side effect)=baselinehazard*exp((β1*radiomimetic agent-induced T lymphocyteapoptosis)+β2*(first clinical parameter, preferably adjuvanthormonotherapy)+β3*(second clinical parameter, preferably tobaccosmoking habits)+ . . . βn*(Clinical parameter (n−1) or biochemicalmarker with n superior or equal to 3), where the baseline hazardcorresponds to the hazard of experiencing the event (ionizingradiation-induced side effect) when all covariates are zero.

In another embodiment, the multivariate Cox model is:

Hazard (experiencing a ionizing radiation-induced side effect)=baselinehazard*exp((β1*radiomimetic agent-induced T lymphocyteapoptosis)+β2*(adjuvant hormonotherapy)+β3*(tobacco smoking habits),where the baseline hazard corresponds to the hazard of experiencing theevent (ionizing radiation-induced side effect) when all covariates arezero.

The right-hand side of the above equation specify the underlyingfunction of the model. The left-hand side of the equation is thepredicted probability that may be presented in a nomogram andcommunicated to the breast cancer patient. Beta coefficients must beestimated for each covariate and converted to hazard ratios as a measureof effect, as in any statistical report. To obtain the predictedprobability of the event in question (experiencing a ionizingradiation-induced side effect), the above equation is calculated using apatient's individual characteristics and the model-derived betacoefficients.

In one embodiment, the baseline hazard is a constant corresponding tothe basal risk to develop a ionizing radiation-induced side effect,without any co-variables. During modelling according to Cox regressionmodel, this baseline hazard may be determined from data coming from areference population as disclosed above.

Clinical parameters ‘1’ to ‘n’ may be selected in the list of clinicalparameters or disease parameters or ionizing radiation treatmentparameters as disclosed hereinabove. In one embodiment, said clinicalparameters are selected from the list comprising age, breast volume,adjuvant hormonotherapy, boost (complement dose of irradiation), nodeirradiation, and tobacco smoking. In one embodiment, said clinicalparameters include adjuvant hormonotherapy and tobacco smoking habits.

In one embodiment, Hazard (experiencing a ionizing radiation-inducedside effect)=baseline hazard*exp((β1*radiomimetic agent-induced Tlymphocyte apoptosis)+β2*(first clinical parameter, preferably adjuvanthormonotherapy)+β3*(second clinical parameter, preferably tobaccosmoking habits)+ . . . βn*(Clinical parameter (n−1) or biochemicalmarker with n superior or equal to 3), where the baseline hazardcorresponds to the hazard of experiencing the event (ionizing radiationinduced side effects, preferably breast late side effect) when allcovariates are zero.

In one embodiment, the value entered for a given parameter in theformula hereinabove is 0 if said parameter is absent and 1 if saidparameter is present (such as, for example, for the presence or absenceof an adjuvant hormonotherapy).

In another embodiment, the value entered for a given parameter in theformula hereinabove corresponds to the measured value of said parameter(such as, for example, for the radiomimetic agent-induced T lymphocyteapoptosis).

In one embodiment, Hazard (experiencing a ionizing radiation-inducedside effect)=baseline hazard*exp(β1*radiomimetic agent-induced Tlymphocyte apoptosis)+β2*(adjuvant hormonotherapy [0=no; 1=yes])+,β3*(tobacco smoking [0=no; 1=yes]) where the baseline hazard correspondsto the hazard of experiencing the event (ionizing radiation induced sideeffects, preferably breast late side effect) when all covariates arezero.

In one embodiment, Hazard (experiencing a ionizing radiation-inducedside effect) may also be named risk to develop a ionizingradiation-induced side effect, for a breast cancer subject.

In one embodiment, based on this multivariate Cox function, the skilledperson would be able to introduce any additional relevant biochemicalmarker(s) and/or clinical parameter(s) to said multivariate Cox model.

In one embodiment, the different coefficients used for the valuesobtained for the different markers in the function of the invention,preferably in the multivariate Cox regression can be calculated throughstatistical analysis in a reference population of patients.

In one embodiment, the method of the invention thus comprises measuringan end-value, wherein said end-value is indicative of the risk of thesubject to develop a ionizing radiation-induced side effect as describedhereinabove. In one embodiment, said risk is estimated taken intoaccount a basal risk (baseline characteristics) and co-variables(biochemical markers and clinical parameters, which are combined in amathematical function).

Therefore, in one embodiment, the “end-value” is the predictedprobability of occurrence of a ionizing radiation-induced side effectfor each subject.

In one embodiment, depending on the end-value obtained for a subject, itis possible to predict for said subject the risk of developing aionizing radiation-induced side effect, during follow-up after ionizingradiation treatment, such as, for example, 3 months or 6, 12, 18, 24, 30or 36 months after the end of the ionizing radiation treatment. Forexample, in one embodiment, an end-value of 92% means an 8% risk todeveloping a ionizing radiation-induced side effect, during ionizingradiation and/or during the follow-up after ionizing radiation, such as,for example, 1 month, 3, 6, 12, 18, 24, 30 or 36 months after the end ofthe ionizing radiation.

In one embodiment, the method of the invention comprises comparing theend-value obtained for a subject with a reference end-value.

In one embodiment, the reference end-value corresponds to the end-valuemeasured in a reference population of breast cancer patients. In oneembodiment, the reference end-value was measured in a referencepopulation comprising breast cancer patients, treated with ionizingradiation and having experienced ionizing radiation-induced side-effectsduring follow-up (such as, for example, during ionizing radiation and/orduring the follow-up after ionizing radiation, such as, for example, 1month, 3, 6, 12, 18, 24, 30 or 36 months after the end of the ionizingradiation). In another embodiment, the reference end-value was measuredin a reference population comprising breast cancer patients, treatedwith ionizing radiation and having experienced no ionizingradiation-induced side-effects during follow-up (such as, for example,during ionizing radiation and/or during the follow-up after ionizingradiation, such as, for example, 1 month, 3, 6, 12, 18, 24, 30 or 36months after the end of the ionizing radiation).

A reference end-value can be derived from population studies, includingwithout limitation, such subjects having similar age range, subjects inthe same or similar ethnic group, similar breast cancer history, similarionizing radiation treatment and the like.

In one embodiment, the reference value is constructed using algorithmsand other methods of statistical and structural classification.

In one embodiment, the reference end-value corresponds to the meanend-value measured in the reference population. In one embodiment of theinvention, the reference end-value corresponds to the median end-valuemeasured in the reference population.

In one embodiment, the method of the invention is computerized (orcomputer-implemented).

In one embodiment, the method of the invention comprises determining ifthe end-value is superior to the reference end-value, or inferior orequal to said reference end-value.

In another embodiment, the method of the invention comprises determiningthe percentile wherein the end-value measured for the subject may bepositioned. According to this embodiment, the end-value measured in areference population are classified in percentiles, wherein theend-values obtained for all subjects of the reference population areranged according to their numerical value in ascending order. In oneembodiment of the invention, the percentiles are percentiles ofsubjects, i.e., each percentile comprises the same number of subjects.Therefore, the first percentile corresponds to subjects with the lowestend-values, while the last percentile corresponds to subjects with thehighest end-values. In one embodiment, when three percentiles are drawn,each percentile is named a tertile. In another embodiment, when fourpercentiles are drawn, each percentile is named a quartile. In anotherembodiment, when five percentiles are drawn, each percentile is named aquintile.

The skilled artisan knows how to determine the reference end-values fromradiomimetic agent-induced T cell apoptosis values obtained in areference population. A non-limiting example of such method include thedrawing of a ROC curve to determine the cut-off of end-value measuredfor the subject with side effect vs subject without side effect (AUROC)which maximize Se and Sp.

In one embodiment, determining the end-value for a breast cancerpatient, will help the physician to adapt the dose and sequences ofionizing radiation treatment to the patient to limit the breast lateside effects, and optionally to adapt the treatment by replacingionizing radiation treatment with other therapeutic treatments.

In a particular embodiment, the end-value (for example obtained with amultivariate Cox function) is used to choose a suitable treatment forthe patient, such as an appropriate ionizing radiation regimen, or tochoose between a mastectomy or conserving surgery.

The volume of irradiation and the prescription dose may thus bediscussed according to the level of risk. In one embodiment, if theend-value is superior to a reference value, there is a risk ofdeveloping a ionizing radiation side effect (preferably a breast lateside effect) after ionizing radiation treatment. In one embodiment, thereference value ranges from about 85 to about 95, such as, for example,is of about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95. Absence ofboost ionizing radiation therapy, absence of node irradiation and doseper fraction less than 2.5 Gy will be different treatment possibilitiesin case of high risk of ionizing radiation side effect (preferably abreast late side effect) and low risk of recurrences of optimal clinicalbenefit.

In one embodiment, the end value is used to choose a suitable treatmentfor the breast cancer patient, such as an appropriate ionizing radiationtherapy dosage regimen, wherein:

-   -   if the patient presents a risk to develop breast late side        effect, the appropriate ionizing radiation dosage regimen will        be decreased (such as, for example by delivery of partial breast        hypofractionated treatment);    -   if the patient presents low risk or no risk to develop breast        late side effect, the appropriate ionizing radiation dosage        regimen may be increased (such as, for example by delivery of        hypofractionated treatment (e.g., 5 or 16 fractions, which are        the common numbers of fractions in such treatments)).

In one embodiment, a patient with a risk to develop a breast late sideeffect ranging from about 5 to about 15% (i.e., with an end-valueranging from about 85 to about 95%), such as, for example, a patientwith a risk of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15% (i.e.,with an end-value of respectively about 95, 94, 93, 92, 91, 90, 89, 88,87, 86 or 85%), the breast cancer patient is considered to be a (high)risk patient.

In one embodiment, a patient with a risk to develop a breast late sideeffect is less than about 5% (i.e., with an end-value superior to about95%), such as, for example, a patient with a risk of about 1, 2, 3, 4,or 5% (i.e., with an end-value superior, respectively, to about 99, 98,97, 96 or 95%), the breast cancer patient is considered to be a low riskpatient.

In another embodiment, the end value is used in the decision ofperforming an immediate breast reconstruction after conserving surgeryor mastectomy. In one embodiment, if said end value ranges from about 85to about 95%, such as, for example, of about 95, 94, 93, 92, 91, 90, 89,88, 87, 86 or 85%, an immediate breast reconstruction after conservingsurgery or mastectomy would be considered.

Another object of the invention is a method for implementing an adaptedpatient care for a breast cancer patient, wherein said method comprises:

-   -   assessing the risk for said patient to develop ionizing        radiation-induced side-effects, using the in vitro method as        described hereinabove;    -   implementing an adapted patient care depending on the risk for        the patient to develop ionizing radiation-induced side-effects.

In one embodiment, the patient is a high risk patient and the adaptedpatient care may be selected from the group comprising decreasedionizing radiation dosage regimen, mastectomy, absence of boost ionizingradiation therapy, absence of node irradiation and dose per fractionless than 2.5 Gy.

In one embodiment, the patient is a low risk patient and the adaptedpatient care may be selected from the group comprising increasedionizing radiation dosage regimen (such as, for example by delivery ofhypofractionated treatment), and immediate breast reconstruction afterconserving surgery or mastectomy.

Another object of the present invention is thus a computer software forimplementing the method of the invention.

In one embodiment, the in vitro method of the invention is implementedwith a microprocessor comprising a software configured to calculate anend-value resulting from the combination of the measures of radiomimeticagent-induced T lymphocyte apoptosis and at least two clinicalparameters (preferably adjuvant hormonotherapy and tobacco smokinghabits), and optionally at least one biochemical marker.

In one embodiment, the in vitro method of the invention is implementedwith a microprocessor comprising a software configured to calculate anend-value resulting from the combination of the measures of radiomimeticagent-induced T lymphocyte apoptosis and at least two clinicalparameters (preferably adjuvant hormonotherapy and tobacco smokinghabits), and optionally at least one biochemical marker.

Another object of the present invention is directed to a systemincluding a machine-readable memory, such as a computer and/or acalculator, and a processor configured to compute said mathematicalfunction, in particular said multivariate Cox function. This system maybe dedicated to perform the method according to the invention.

In particular embodiment, said system comprises additionally a modulefor executing a software to build a nomogram (linear predictor between0-100 for each parameter including main effect, interaction andpiecewise linear effect) and calculate the risk (corresponding to theend-value) for the subject to develop a ionizing radiation-inducedside-effect.

In one embodiment, the in vitro method of the invention thus comprises:

-   -   contacting T cells with at least radiomimetic agent for all        subjects of a reference population and measuring radiomimetic        agent-induced T cells apoptosis    -   optionally measuring at least one biochemical marker for all        subjects of the reference population    -   measuring at least two clinical parameters, preferably adjuvant        hormonotherapy and tobacco smoking, for all subjects of the        reference population;    -   univariate analysis (estimation for each parameter one by one in        order to select all significant parameters with p-value≤0.2)        under a Cox regression model;    -   multivariate analysis (estimation including all selected        parameters by univariate analysis+adding optional        non-significant parameters which are clinically relevant) under        Cox regression model;    -   selection of significant parameters and/or clinically relevant        parameters to obtain the final model whose linear predictor were        extracted to estimate the risk (probability) to develop a        ionizing radiation-induced side effect; linear predictor was        integrated in a software;    -   execution of a software to build a nomogram according to Iasonos        et al. 2008 (linear predictor between 0-100 for each parameter        including main effect, interaction and piecewise linear effect).        This representation thus gives the risk (probability) of        developing a ionizing radiation-induced side effect by        calculation of an end-value after ionizing radiation for each        breast cancer patient according to each individual parameter.

Therefore, in one embodiment, the present invention comprisesvisualizing the end-value obtained for the breast cancer subject on anomogram. A nomogram is a popular visual plot to display the predictprobabilities of occurrence of an event for decision support.

In one embodiment, to build this nomogram after fitting the Coxmultivariate model, a linear predictor is obtained according to themethod described by Iasonos et al. (2008). Another object of the presentinvention is thus a user-friendly interface, i.e., a nomogram, computeror calculator, implementing said mathematical combination (preferablysaid multivariate Cox function), to help physician to interpret the riskof developing a ionizing radiation-induced side effect, e.g., a breastlate side effect during and/or after a ionizing radiation treatment.Accordingly, the present invention encompasses a nomogram implementingthe mathematical function of the invention (preferably the multivariateCox function according to the invention).

As used herein, “a nomogram” refers to a graphical representation ofprognosis formula(ae) from a mathematical function as described herein,such as, for example, a multivariate Cox modelling, which allows forestimation of the risk of developing a ionizing radiation-induced sideeffect, in a subject. In one embodiment, said nomogram is based on oneor more readily obtained parameters, including, but not limited to,radiomimetic agent-induced T cell apoptosis, adjuvant hormonotherapy,and tobacco smoking.

The usefulness of a nomogram is that it maps the predicted probabilitiesinto points on a scale from 0 to 100 in a user-friendly graphicalinterface. The total points accumulated by the various covariatescorrespond to the predicted risk for a patient.

According to one embodiment, the method of the invention comprisesimplementing the data obtained at step b), at step c) and optionally atstep d) to a computer or a calculator that will calculate themathematical combination (preferably the multivariate Cox regression)and the risk of developing of a ionizing radiation-induced side effect.The data obtained by the physician is therefore more easilyinterpretable, and will allow for an improvement in the process fordeciding the adapted patient care.

Another object of the present invention is a kit for implementing themethod of the invention, in particular for collecting data of a subjectto be further used for detecting the risk of developing of a ionizingradiation-induced side effect in a breast cancer subject using themethod of the present invention, wherein the kit comprises:

-   -   a box/container and bag suited for biological transportation of        a T cell containing sample, in particular a blood sample, and        optionally reagents for isolating T cells;    -   reagents for measuring radiomimetic agent-induced T cell        apoptosis, and optionally at least one biochemical marker as        defined herein; and    -   forms to be completed by the patient and/or the nurse and/or the        physician, specifically designed and necessary to implement the        method of the invention.

As an example, the forms may contain specific questions aimed atcollecting information necessary to run the predictive analysis such as,age, whether the patient has undergone or will undergo an adjuvanttreatment (chemotherapy or hormone therapy, for example), tobacco habitand date and time when the T cell containing sample was recovered.

Another object of the present invention is thus a kit for detecting therisk of developing a ionizing radiation-induced side effect (preferablya breast late side effect), in a subject using the method of the presentinvention, wherein said kit comprises:

-   -   reagents for determining radiomimetic agent-induced T lymphocyte        apoptosis as defined in the present invention;    -   optionally means for collecting information on the at least two        clinical parameters (preferably adjuvant hormonotherapy and        tobacco smoking habits) according to the present invention, such        as a survey;    -   optionally means for measuring at least one biochemical marker        according to the present invention; and    -   optionally a nomogram according to the invention.

In one embodiment, the reagents for determining radiomimeticagent-induced T lymphocyte apoptosis as defined in the present inventioncorrespond to some or all specific reagents required to:

-   -   run the step of culture of T cells as described hereinabove, in        the presence of at least one radiomimetic agent, such as, for        example, a culture medium suitable for culturing T cells, and        said radiomimetic agent (such as, for example, bleomycin);    -   run the apoptosis measurement assay (for example, using a flow        cytometer), such as, for example, propidium iodide, fluorochrome        coupled-annexin, YO-PRO dyes, PO-PRO dyes, Resazurin, Hoechst,        fluorochrome coupled-caspases.

In one embodiment, the reagents for determining the values of at leastone biochemical marker according to the invention correspond to some orall specific reagents required to run the proteins and/or genesbiosensitivity measurement assay in an independent laboratory.

In one embodiment, the means for collecting information of at least twoclinical parameters according to the invention correspond to specificforms to be completed by the patient and/or the nurse and/or thephysician, specifically designed and required to run the method of theinvention and the nomogram analysis. In a preferred embodiment, theseforms may contain specific questions aimed at collecting informationnecessary to run the predictive analysis such as whether the patient hasundergone or will undergo an adjuvant treatment (chemotherapy,hormonotherapy, for example), and tobacco habit.

Examples

The present invention is further illustrated by the following examples.

Materials and Methods

Blood Sample

Blood samples were purchased from Etablissement Français du Sang (EFS)with the convention number 21PLER2016-0100 AV01. Blood samples werecollected from healthy donors in 5 mL heparinized tubes withoutseparation gel.

Culture Conditions

Blood cells were grown in RPMI-1640 with glutamax (Fisher scientific,France) supplemented with 20% fetal calf serum (FCS) (EuroBio, France).Cells were maintained at 37° C. with 5% CO₂ during the experiments.

Products

Bleomycin (EuroMedex) was solubilized in sterile MilliQ water at 50mg/mL according to manufacturer instructions and stored at −80° C.

Radiation-Induced CD8 T-Lymphocyte Apoptosis (RILA) Procedure

The protocol was adapted from studies of Ozsahin et al. (Ozsahin,Crompton et al. 2005).

Before radiotherapy (RT), one blood sample was collected from eachpatient in a 5-mL heparinized tube. Twenty or twenty-four hours afterblood collection, 200 μl of blood were aliquoted into a 6-well platecontaining 2 mL of RPMI-20% FCS. All tests were carried out intriplicate for both 0 and 8 Gy. Ex-vivo irradiations were deliveredafter 24 hours of blood cell culture using the Xenx irradiator platform(XStrahl, UK). Then, the plates were immediately incubated for 48 hoursat 37° C. (5% CO₂).

Samples were then centrifuged for 5 min at 300 g. The pellets wereresuspended in phosphate-buffered saline (PBS) containing 10 μl ofanti-human CD8-FITC antibody (Becton Dickinson, USA) and incubated for20 minutes at room temperature. Then, red blood cells were lysed byaddition of 4 ml of lysis buffer diluted 1:10 in water (BectonDickinson, USA). After another 20 minutes incubation time at roomtemperature, samples were centrifuged for 5 min at 300 g and the pelletswere washed with 3 ml of PBS. Pellets were suspended in 200 μl of PBScontaining 25 μg/ml of propidium iodide (Sigma, France) and 5 μl ofRNAse A at 10 mg/ml (Qiagen, France). The samples were analyzed by flowcytometry within the next hours, using the CytoFlex (Beckman Coulter,USA).

Bleomycin-Induced CD8 T-Lymphocyte Apoptosis Procedure

Irradiation and Treatment on Blood Samples

Different irradiation doses (0 Gy, 6 Gy, 8 Gy and 10 Gy) and differentBleomycin concentrations (50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, 250μg/mL) were applied to aliquots of the same blood sample.

Irradiations and culture were performed as previously described. Thestaining was performed 48 hours after the irradiation using the sameprotocol as previously described.

For the Bleomycin treatment, 2 protocols were tested:

-   -   Treatment 24 hours after blood cell culture

Two hundred microliters of blood were aliquoted into a 6-well platecontaining 2 mL of RPMI-20% FCS, about 24 hours after blood collection.Bleomycin treatment, at the different concentrations, was performed 24hours after blood cell culture by adding the Bleomycin directly into thewells. The staining for detecting apoptosis was performed as describedabove and 48 hours after the bleomycin treatment (thus, after less than50 hours of contact of bleomycin with the cells).

-   -   Treatment during blood cell culture:

Two hundred microliters of blood were aliquoted into a 6-well platecontaining 2 mL of RPMI-20% FCS-bleomycin, about 24 hours after bloodcollection. The cell culture media containing the differentconcentrations of bleomycin were made fresh just before starting thecell culture. After bleomycin incubation with blood cells for at least50 hours (generally more than about 60 hours, in particular between 65and 75 hours), samples were stained for flow cytometry analysis asdescribed above.

Statistical Analysis

Two mixed linear models with fixed intercept, fixed slope and randomintercept were used to model the RILA rate evolution according to thecompound or to the irradiation dose in Gray.

Analysis were performed with the R software (version 3.3.1)

Results

Prolonged Bleomycin Treatment Induces a Similar T Lymphocyte ApoptosisRate than an 8 Gy Irradiation

The aim of this study is to determine the bleomycin conditions of use toobtain a similar T lymphocyte apoptosis rate than an 8 Gy irradiation.

Two treatment conditions were tested on the same blood sample: bloodcells were treated by several bleomycin concentrations 24 hours aftercell culture or from the start of cell culture. A control experiment wasperformed by irradiating blood samples at 8 Gy.

The results described in the Table 1 are representative of allexperiments. Blood samples were treated by a concentration of bleomycinbetween 50 μg/mL and 500 μg/mL. When the treatment was performed 24hours after blood cell culture, no bleomycin concentration, applied forless than 50 hours, induces the apoptosis rate observed after 8 Gyirradiation.

In contrast, when treatment was applied at the start of blood cellculture, the apoptosis rate obtained after irradiation was achieved witha bleomycin concentration lower than 300 μg/ml. These results show thatthe treatment must be performed during at least 50 hours (such as, forexample, with a treatment starting with the cell culture) to obtain asimilar T lymphocyte apoptosis rate than an 8 Gy irradiation. Withoutwilling to be bound to any theory, it is suggested that bleomycin mayneed more time to induce the same T lymphocyte apoptosis rate as an 8 Gyirradiation. It is to be noted that the duration of contact of thebleomycin with the cells may vary according to the concentration of thebleomycin, but should be long enough (i.e. more than 50 hours) in orderto be able to reproduce the apoptosis level observed in RILA.

However, it is preferred to use low concentrations of bleomycin, as itis an expensive compound.

TABLE 1 Comparison of bleomycin treatment on the same blood sampleapplied during cell culture or 24 hours later. 8 Gy irradiation Tlymphocytes apoptosis rate (%) (%) 28.2 Bleomycin From the start 24hours treatment of cell culture (%) after cell culture (%) 50 μg/mL12.47 3.98 100 μg/ml 19.14 2.72 200 μg/ml 23.44 10.13 300 μg/ml 26.3211.9 400 μg/ml 30.68 16.78

This table clearly shows that the exposition of the bleomycin to thecells from the start of cell cultures (thereby prolonging the expositionof the cells to the compound) leads to a higher level of apoptosis ofthe T lymphocytes cells than when bleomycin is added from 24 hours afterstart culture. One can see that the prolongation of exposure leads to agreat increase of apoptosis, whatever the concentration of bleomycin.

Identification of a Correlation Between Bleomycin Treatment andIrradiation

In order to characterize the correlation between bleomycin treatment andirradiation, different bleomycin concentrations and differentirradiation doses were applied on the same blood sample. The Tlymphocytes apoptosis rate seems to respond in a linear way according tothe irradiation dose (Gy) and according to the bleomycin concentration.(Data not shown).

Apoptosis rate model according to the irradiation (Gy):y _(i) ^(ir) =f _(i) ^(ir)(x ^(ir))=β₀+β₁ x ^(ir) +b _(0i)+ϵ_(i)  (1)

Apoptosis rate model according to the compound (μg/mL):y _(i) ^(co) =f _(i) ^(co)(x ^(co))=α₀+α₁ x ^(co) +a _(0i)+ϵ_(i)  (2)

The combination of the two models hereinabove allows determining a doseof bleomycin concentration ({tilde over (x)}^(co)) to be applied formimicking the effect of an irradiation (x^(ir)):

${\overset{\sim}{x}}^{co} = \frac{{\overset{\hat{}}{\beta}}_{0} + {x^{ir}{\overset{\hat{}}{\beta}}_{1}} - {\overset{\hat{}}{\alpha}}_{0}}{{\overset{\hat{}}{\alpha}}_{1}}$

Using RILA measures in 10 donors, it was possible to estimate thedifferent parameters of this mathematic formula. The parameters obtainedby such formula can be calculated for given duration of exposition andconcentration of bleomycin.

{circumflex over (β)}₀ ranges from about 10 to about 20. This parametercorresponds to an apoptosis average value for irradiation level at zero.

{circumflex over (β)}₁ ranges from about 0.5 to about 2. This parametercorresponds to the slope, i.e., to the increase of apoptosis for each 1Gy increase.

{circumflex over (α)}₀ ranges from about 7.5 to about 20. This parametercorresponds to the apoptosis average value for bleomycin concentrationat zero.

{circumflex over (α)}₁ ranges from about 0.03 to about 0.15. Thisparameter corresponds to the slope, i.e., to the increase of apoptosisfor each 1 μg/mL increase.

Bleomycin Concentration Validation

The Bleomycin concentration determined with the equation disclosedhereinabove was validated on 40 blood samples. For each blood samples, Tlymphocytes apoptosis rate were obtained after an 8 Gy irradiation (RILAprocedure disclosed above) or by treatment with 135-160 μg/mL ofbleomycin, for a duration of exposition comprised between 65 and 75hours (about 70-72 hours).

For a same donor, the apoptosis rates obtained by the two treatmentswere compared by calculating the coefficient of variation (CV). The CVbetween these 2 conditions were calculated according to the followingformula: CV=(Standard deviation/Average apoptosis)×100.

The average CV for these 40 donors was 6.5%. This percentage was lowerthan 10%, which represents the experimental variation of the RILA assay.

These results were later reproduced on a larger (more than 100) numberor donors.

It is also to be noted that the batch of bleomycin used for theseexperiments is different from the one tested above for the determinationof the duration of exposure. Depending on the batch, a variation in theeffective concentration can be observed and the effective concentrationmust be thus verified for any new batch of bleomycin used, in order toadjust the concentration. However, the duration of exposure must stillremain higher than 50 hours.

These results thus show that it is possible to reproduce the apoptosisconsequences of irradiation by using bleomycin as a radiomimetic, andthat the contact of the radiomimetic shall be more than 50 hours.

The invention claimed is:
 1. An in vitro method for mimicking the effectof an ionizing radiation on T cells of a subject, wherein said effect isthe percentage of apoptosis of lymphocytes observed 48 hours afterirradiation, comprising contacting the T cells of the subject with aconcentration of bleomycin for a period of time of 50 hours to 75 hours,and measuring the T cells apoptosis percentage, wherein the T cellsapoptosis percentage after contact with bleomycin corresponds to theapoptosis percentage of T cells 48 hours after being subjected toionizing radiation, wherein the concentration of bleomycin ranges fromabout 100 μg/mL to about 300 μg/mL and wherein the method mimics theeffect of an ionizing radiation at a dose ranging from about 2 Gy toabout 10 Gy.
 2. The in vitro method of claim 1, wherein theconcentration of the bleomycin is determined using the followingmathematic formula${\overset{\sim}{x}}^{co} = \frac{{\overset{\hat{}}{\beta}}_{0} + {x^{ir}{\overset{\hat{}}{\beta}}_{1}} - {\overset{\hat{}}{\alpha}}_{0}}{{\overset{\hat{}}{\alpha}}_{1}}$wherein ({tilde over (x)}^(co)) is the dose of compound to be appliedfor mimicking the effect of a specific irradiation dose (x^(ir)),{circumflex over (β)}₀ corresponds to an apoptosis average value forirradiation level at zero, {circumflex over (β)}₁ corresponds to theslope, i.e., to the increase of apoptosis for each 1 Gy increase,{circumflex over (α)}₀ corresponds to the apoptosis average value forbleomycin concentration at zero and {circumflex over (α)}₁ correspondsto the slope, i.e., to the increase of apoptosis for each 1 μg/mLincrease.
 3. The in vitro method of claim 1, wherein the T cells arecontacted with bleomycin at a temperature of about 37° C. and in thepresence of about 5% CO₂.
 4. The in vitro method of claim 1, wherein theT cells are CD8⁺ T cells.
 5. The in vitro method of claim 1, wherein theT cells are comprised in a T cell containing sample selected from ablood sample, a sample recovered from bone marrow, lymph node tissue,cord blood, thymus tissue, tissue from a site of infection, ascites,pleural effusion, spleen tissue, and a tumor sample.
 6. The in vitromethod of claim 1, wherein the T cells contacted with bleomycin for aperiod of time of at least 50 hours are from a T cell containing samplepreviously obtained from the subject, and wherein the individualradiosensitivity of the subject is determined.
 7. The in vitro method ofclaim 6, wherein the subject is diagnosed with cancer.
 8. The in vitromethod of claim 6, wherein the subject is treated or is planned to betreated with an ionizing radiation treatment.
 9. The in vitro method ofclaim 6, wherein the risk of developing side effects after an ionizingradiation treatment is also determined.
 10. The in vitro method of claim6, comprising: (a) contacting a T cell containing sample previouslyobtained from a subject with bleomycin, and (b) measuring CD8⁺ T cellapoptosis in the sample from the subject.
 11. The in vitro method ofclaim 1, wherein the method mimics the effect of a ionizing radiation ata dose of about 8 Gy.
 12. The method of claim 5, wherein the T cellcontaining sample is a blood sample or a whole blood sample.
 13. An invitro method for mimicking the effect of an ionizing radiation at a doseof 8 Gy on T cells of a subject, wherein said effect is the percentageof apoptosis of lymphocytes observed 48 hours after irradiation,comprising contacting the T cells of the subject with a concentration ofbleomycin for a period of time of 65 to 75 hours, and measuring the Tcell apoptosis percentage, wherein the T cells apoptosis percentageafter contact with bleomycin corresponds to the apoptosis percentage ofT cells 48 hours after being subjected to the ionizing radiation,wherein the concentration of bleomycin ranges from about 135 μg/mL toabout 160 μg/mL.
 14. The in vitro method of claim 13, wherein bleomycinis applied for 70 to 72 hours.